Abstract
The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.
Highlights
Nucleotide excision repair (NER) processes diverse lesions in DNA damaged by chemical modification or UV radiation [1,2,3,4]
The Walker B mutant E512AUvrA2, in which proximal ATP binding is preserved but hydrolysis is disrupted, exhibits a burst of ATP hydrolysis and Pi release by the wild-type distal sites, followed by the linear steady state, again as seen in the absence of DNA (Figure 6A). These results show that in the UvrA2–native DNA complex, ATP binding to the weak proximal sites remains necessary for ATP hydrolysis by the distal sites, and that native DNA does not fundamentally alter the distal site ATPase mechanism, except to speed up the kcat to some extent
Information from prior biochemical and structural studies, we propose a model of how each ATPase site contributes to UvrA2 function in NER (Figure 10)
Summary
Nucleotide excision repair (NER) processes diverse lesions in DNA damaged by chemical modification (e.g. benzo[a]pyrene adducts) or UV radiation (e.g. cyclobutane pyrimidine dimers) [1,2,3,4]. This multi-step pathway employs different proteins to scan the genome, distinguish damaged from undamaged (native) DNA, incise and remove the lesion-containing section of single-stranded DNA and, mediate DNA synthesis using the undamaged strand as template (Figure 1A). Subsequent strand displacement by UvrD helicase allows gap filling by DNA polymerase I, and DNA ligase completes repair [1]
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